1. Field of the Invention
This invention relates to modified or mutant angiogenin proteins and DNA sequences which encode the modified or mutant proteins. Additionally, the invention relates to vectors, host cells and methods of expression of the modified or mutant angiogenin proteins. In particular, the modified or mutant angiogenin proteins of the present invention are genetically engineered covalent angiogenin/ribonuclease (RNase) hybrids. These hybrids are useful for the identification of structural components of angiogenin necessary for angiogenin's characteristic activities. In particular, regional mutagenesis may be used to generate such angiogenin/RNase hybrid proteins, which are derivatives of angiogenin in which particular regions of primary structure have been replaced with the corresponding segments of RNase. Preparation and characterization of such proteins allows the identification of segments, such as an N-terminal segment according to the present invention, that may be critical to angiogenin's characteristic activities. Specifically, the present invention relates to a novel recombinant hybrid protein with increased angiogenic potency, ARH-III, in which N-terminal residues 8-22 of angiogenin are replaced by the corresponding region (residues 7-21) of RNase via regional mutagenesis. Such recombinant mutant angiogenin proteins may be produced in sufficient quantities to permit their application as therapeutics or diagnostics.
2. Background of the Art
Angiogenesis, the process of developing a hemovascular network, is essential for the growth of solid tumors and is a component of normal wound healing and growth processes. It has also been implicated in the pathophysiology of atherogenesis, arthritis, and diabetic retinopathy. It is characterized by the directed growth of new capillaries toward a specific stimulus. This growth, mediated by the migration of endothelial cells, may proceed independently of endothelial cell mitosis.
The molecular messengers responsible for the process of angiogenesis have long been sought. Greenblatt and Shubik, 1968, J. Natl. Cancer Inst. 41: 111-124, concluded that tumor-induced neovascularization is mediated by a diffusible substance. Subsequently, a variety of soluble mediators have been implicated in the induction of neovascularization. These include prostaglandins (Auerbach, in Lymphokines, Pick and Landy, eds., 69-88, Academic Press, New York, 1981), human urokinase (Berman et al., 1982, Invest. Opthalm. Vis. Sci. 22: 191-199), copper (Raju et al., 1982, J. Natl. Cancer Inst. 69: 1183-1188), and various "angiogenesis factors."
A variety of angiogenesis factors have been derived from tumor cells, wound fluid (Banda et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7773-7777; Banda et al., U.S. Pat. No. 4,503,038), and retinal cells (D'Amore, 1981, Proc. Natl. Acad. Sci. USA 78: 3068-3072). Tumor-derived angiogenesis factors have in general been poorly characterized. Folkman et al., 1971, J. Exp. Med. 133: 275-288, isolated tumor angiogenesis factor from the Walker 256 rat ascites tumor. The factor was mitogenic for capillary endothelial cells and was inactivated by RNase. Tuan et al., 1973, Biochemistry 12: 3159-3165, found mitogenic and angiogenic activity in the nonhistone proteins of the Walker 256 tumor. The active fraction was a mixture of proteins and carbohydrate. A variety of animal and human tumors have been shown to produce angiogenesis factor(s) (Phillips and Kumar, 1979, Int. J. Cancer 23: 82-88, but the chemical nature of the factor(s) was not determined. A low molecular weight non-protein component from Walker 256 tumors has also been shown to be angiogenic and mitogenic (Weiss et al., 1979, Br. J. Cancer 40: 493-496). An angiogenesis factor with a molecular weight of 400-800 daltons was purified to homogeneity by Fenselau et al., 1981, J. Biol. Chem. 256: 9605-9611, but it was not further characterized. Human lung tumor cells have been shown to secrete an angiogenesis factor comprising a high molecular weight carrier and a low molecular weight, possibly non-protein, active component (Kumar et al., 1983, Int. J. Cancer 32: 461-464). Vallee et al., 1985, Experientia 41: 1-15, found angiogenic activity associated with three fractions from Walker 256 tumors. Tolbert et al. (U.S. Pat. No. 4,229,531) disclose the production of angiogenesis factor from the human adenocarcinoma cell line HT-29, but the material was only partially purified and was not chemically characterized. Isolation of genes responsible for the production of the above described angiogenesis factors has not been reported at least in part due to the lack of purity and characterization of the factors.
Isolation of angiogenesis factors has employed a number of different techniques, including: high performance liquid chromatography (Banda et al., supra); solvent extraction (Folkman et al., supra); chromatography on silica gel (Fenselau et al., supra); DEAE cellulose (Weiss et al., supra), or Sephadex (Tuan et al, supra); and affinity chromatography (Weiss et al., supra).
Vallee et al. (U.S. Pat. No. 4,727,137, which is hereby incorporated by reference) have purified an angiogenic protein from a human adenocarcinoma cell line. This protein has been identified in normal human plasma (Shapiro, et al., 1987, Biochem. 26: 5141-5146). The purified protein, known as angiogenin, was chemically characterized and its amino acid sequence determined. Two distinct activities have been demonstrated for the human tumor-derived angiogenin. First, it was reported to behave as a very potent angiogenic factor in vivo (Fett et al., 1985, Biochem. 24: 5480-5486). Second, it has been found to exhibit a characteristic ribonucleolytic activity toward 28S and 18S rRNA that differs significantly from that of pancreatic RNase in two respects: (a) it requires up to 10.sup.5 as much angiogenin to obtain the same degree of rRNA degradation as with RNase; and (b) the products are much larger, i.e., from 100 to 500 nucleotides; in addition, it is essentially inactive toward classic RNase A substrates. (Shapiro et al., 1986, Biochem. 25: 3527-3532; St. Clair et al., 1987 , Proc. Natl. Acad. Sci. USA 84: 8330-8334).
In addition, Vallee et al. (U.S. Pat. No. 4,721,672, which is also hereby incorporated by reference) have cloned the gene (both cDNA and genomic) encoding the angiogenic protein claimed in U.S. Pat. No. 4,727,137 from a human liver cDNA library and a human genomic library. The angiogenin gene was cloned into vectors and the recombinant vectors encoding the angiogenin gene were used to transform or transfect host cells. Such transformed or transfected cells express a human angiogenin protein.
Based on the sequence of the angiogenin gene described and claimed in U.S. Pat. No. 4,721,672, several groups have prepared synthetic angiogenin genes. Denefle et al., 1987, Gene 56: 61-70, prepared a synthetic gene coding for human angiogenin, which was designed to use codons found in highly expressed E. coli proteins. The synthetic gene was ligated into a pBR322-derived expression vector constructed to contain the E. coli tryptophan (trp) promoter. This E. coli-produced angiogenin was found to be insoluble but could be easily renatured and purified. The purified angiogenin exhibited angiogenic activity and ribonucleolytic activity similar to that described for natural angiogenin purified by Vallee et al. (U.S. Pat. No. 4,727,137) from human adenocarcinoma cells. A different synthetic gene for angiogenin was prepared by Hoechst (German Patent Application P3716722.7) encoding a leucine at amino acid position 30 instead of methionine as found in the natural (i.e. wildtype) angiogenin gene described and claimed in U.S. Pat. No. 4,721,672. This synthetic Leu-30 angiogenin gene was designed to use codons preferentially expressed in E. coli . The gene was subcloned into a vector containing a modified trp promoter (European Patent Application 0198415) and a translation initiation region (TIR) sequence (Gene 41: 201-206, 1986; EMBO J. 4: 519-526, 1985) to increase translation efficiency. The synthetic gene is thus under direct control of the trp promoter and expression is induced by addition of indole-3-acrylic acid or by tryptophan starvation. The Leu-30 angiogenin protein could be purified and was found to exhibit angiogenic and ribonucleolytic activity similar to that of the wildtype (Met-30) angiogenin.
All the human angiogenin proteins just described, whether plasma-derived, tumor cell-derived or recombinant DNA-derived (cDNA, genomic DNA or synthetic DNA) exhibit both angiogenic activity and ribonucleolytic activity. Indeed, one of the most intriguing features of angiogenin is its structural homology with mammalian pancreatic RNases. This structural relationship should permit the study of the mechanism of action of angiogenin, as well as the relationship between the angiogenic and enzymatic (i.e. ribonucleolytic) activities of angiogenin.
In vivo, angiogenin is a potent inducer of blood vessel growth as measured by the chick chorioallantoic membrane (CAM) and rabbit cornea assays (Fett et al., 1985, Biochemistry 24: 5480-5486). In vitro, angiogenin induces multiple responses in endothelial cells including activation of phospholipase C and secretion of prostacyclins (Bicknell & Vallee, 1988, Proc. Natl. Acad. Sci. USA 85: 5961-5965; Bicknell & Vallee, 1989, Proc. Natl. Acad. Sci. USA 86: 1573-1577); it also inhibits cell free protein translation by specific cleavage of 18S RNA within the 40S ribosomal subunit (St. Clair et al., 1987, Proc. Natl. Acad. Sci. USA 84: 8330-8334; St. Clair et al., 1988, Biochemistry 27: 7263-7268).
Angiogenin is structurally homologous to the pancreatic RNase family of enzymes, having 34% sequence identity to the most extensively studied member of this group, bovine pancreatic RNase A (RNase A) (Strydom et al., 1985, Biochemistry 24: 5486-5494; Kurachi et al., 1985, Biochemistry 24: 5494-5499). Angiogenin's tertiary structure is similar to that of RNase A as well, based on (i) conservation of three of four disulfide bonds, (ii) extremely tight binding to placental ribonuclease inhibitor (PRI) (Shapiro & Vallee, 1987, Proc. Natl. Acad. Sci. USA 84: 2238-2241; Lee et al., 1989b, Biochemistry 28: 225-230; Blackburn et al., 1977, J. Biol. Chem 252: 5904-5910), and (iii) a computer-generated three-dimensional structure (Palmer et al., 1986, Proc. Natl. Acad. Sci. USA 83: 1965-1969). The three essential catalytic residues of RNase A (His-12, Lys-41, and His-119) are conserved in angiogenin (His-13, Lys-40, and His-114) as are many other key active site and structural residues. Indeed, as stated above, angiogenin does possess ribonucleolytic activity, but of a type far different from other RNases. Angiogenin's activities toward most conventional substrates are five to six orders of magnitude below those of RNase A (Shapiro et al., 1987, Proc. Natl. Acad. Sci. USA 84: 8783-8787; Shapiro et al., 1988, Anal. Biochem. 175: 450-461; Harper & Vallee, 1989, Biochemistry 28: 1875-84). Thus, despite the structural similarities, the vast differences in in vivo and in vitro activities of angiogenin compared to the RNases (which have not been shown to induce angiogenesis or second messenger activities) are clearly indicative of a distinct physiological function for angiogenin.
Recent studies have shown that the active-site histidine and lysine residues of angiogenin are required for both angiogenic and ribonucleolytic activities (Shapiro et al., 1986, Biochemistry 25: 3527-3532; Shapiro et al., 1987b, Proc. Natl. Acad. Sci. U.S.A. 84: 8783-8787; Shapiro et al., 1988b, Biochem. Biophys. Res. Commun. 156: 530-536; Shapiro et al., 1989, Biochemistry 28: 1726-32; and Shapiro and Vallee, 1989, Biochemistry 28: 7401-8). However, additional molecular features must be critical to angiogenesis, since bovine pancreatic RNase A contains the corresponding histidine and lysine residues yet it is not angiogenic. Such additional molecular features, if they could be identified, should include individual residues and/or regions of sequence which are unique to angiogenin. Harper and Vallee, 1989, Biochem. 28: 1875-84 have recognized that one striking structural difference between angiogenin and RNase is the virtual absence of sequence similarity within the region of RNase that contains the Cys-65-Cys-72 disulfide bond. Angiogenin lacks this disulfide linkage. Having identified this region of angiogenin, Harper and Vallee, 1989, supra, have described a novel genetically-engineered covalent angiogenin/RNase hybrid protein, ARH-I, wherein the segment of angiogenin comprising amino acid residues 58-70 was replaced by the corresponding segment (residues 59-73) of RNase A. The genetically-engineered replacement of this segment of angiogenin by the corresponding segment from RNase A resulted in a covalent hybrid protein with characteristics more like RNase A. ARH-I exhibited dramatically increased RNase-like enzymatic activity but a markedly decreased angiogenic biological activity. Harper and Vallee, 1989, supra, were unable to prepare or identify a hybrid protein with increased angiogenic biological activity. Such a hybrid would be unexpected in view of their experimental design to insert an RNase-unique region into angiogenin.